1 IMMUNOMODULATING TREATMENT WITH LOW DOSE

JOURNAL OF BIOLOGICAL REGULATORS & HOMEOSTATIC AGENTS
Vol. 28, no. 1, 0-0 (2014)
IMMUNOMODULATING TREATMENT WITH LOW DOSE INTERLEUKIN-4,
INTERLEUKIN-10 AND INTERLEUKIN-11 IN PSORIASIS VULGARIS
M.L. ROBERTI1, L. RICOTTINI2, A. CAPPONI3, E. SCLAUZERO4, P. VICENTI5,
E. FIORENTINI6, C. SAVOIA7, G. SCORNAVACCA8, D. BRAZIOLI9, L. GAIO10,
R. GIANNETTI11, C. IGNAZZI12, G. MELONI13 and L.M. CHINNI14
Private Practice, Rome, Italy; 2Centro medico polispecialistico “Sinergheia”,
Rome, Italy; 3Private Practice, Latina, Italy; 4OSTEMDA, Organizzazione Strategie Terapeutiche
Empowerment e Metodologie Diagnostiche Avanzate, Udine, Italy; 5Private Practice, Altamura,
Bari, Italy; 6Ambulatorio di dermatologia, Aversa, Caserta, Italy; 7Private Practice, Fino Mornasco,
Como, Italy; 8Private Practice, Catania, Italy; 9Private Practice, Torino, Italy; 10Private Practice,
Caserta, Italy; 11Poliambulatorio di medicina convenzionata“Aurelia”, Rome, Italy; 12Azienda
Sanitaria Locale, Putignano, Bari, Italy; 13Poliambulatorio “GEA Medica”, Montebelluna, Treviso,
Italy; 14Istituto Dermopatico dell’Immacolata, Roma, Italy
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Psoriasis is a chronic inflammatory skin disease affecting approximately 2-3% of the world population;
it is characterised by hyperproliferation and hyperplasia of the superficial layers of the epidermis.
Inappropriate signals released by the immune system determine an altered keratinocyte differentiation,
resulting in the formation of desquamating, thickened, inflamed and erythematous plaques. The aim of
this investigation was to study the pharmacological activity and safety of three low dose cytokines, GunaInterleukin 4, Guna-Interleukin 10 and Guna-Interleukin 11 at the concentration of 10 fg/ml in patients
affected by moderate to slight psoriasis vulgaris. The multicenter, double-blind, randomized, placebocontrolled clinical trial involved 48 patients who were enrolled and followed up according to a 8-month
experimental project. All patients received, according to a cross-over model, either the experimental
treatment or placebo, alternatively. Globally, in the 41 evaluated patients it was observed a PASI significant
reduction (Friedman test: p=0.00960). The DLQI too decreased significantly in all subjects compared to
baseline (Friedman test: p=0.00007). The safety of the treatment with three low dose cytokines administered
simultaneously was proved; no adverse event was reported during the whole trial.
P
Psoriasis is a common chronic inflammatory
skin disease, most often characterized by thickened
erythematous scaly plaques, and appears in a variety
of forms with distinct characteristics. The most
common form, psoriasis vulgaris, affects 1-3% of
the Caucasian population, usually persists, with 40%
developing seronegative arthritis, and has a very
negative impact on quality of life (1).
The existence of familial cases of psoriasis and
the occurrence of the pathology in monozygotic
twins demonstrates the role of genetic factors. The
mode of inheritance of psoriasis is complex. In the
multifactorial hypotesis environmental triggering
factors (traumas, infections, stress, climate)
interplays with genetic factors (2).
The pathogenesis of disease is still not clear,
therefore psoriasis is generally regarded as a T-cell
mediated autoimmune disease. Although psoriasis
Key words: Psoriasis vulgaris, interleukin-4, interleukin-10, interleukin-11, PASI score, DLQI
Mailing address:
Maria Luisa Roberti MD,
Private Practitioner,
Piazza di Villa Carpegna, 42/B,
00165 Roma, Italy
Tel.: +39 06 6634750
e-mail: [email protected]
0393-974X (2014)
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Unauthorized reproduction may result in financial and other penalties
DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF
INTEREST RELEVANT TO THIS ARTICLE.
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M.L. ROBERTI ET AL.
was initially classified as a Th-1-polarized disease,
a clear role for CD4+ T cells that produce IL-2,
IL-6, IL-17 and IL-22 has been demonstrated. The
cytokines show a key role in the development and
maintenance of psoriasic lesions. The hypothesis of
a cytokine network in psoriasis proposes a central
role of pro-inflammatory cytokines, including
TNF-α. In this disease, three predominant cytokines
seem to be at play: type I interferons, IFN-γ and
TNF-α. In psoriasis, IFN-γ, the prototype Th-1
cytokine, interplays with TNF-α, IL-17, IL-22,
and IL-6 to inflammatory process (3, 4). Therapies
targeting T cells seem to be very effective in the
treatment of skin lesions and arthritis involvement.
Strategies for intervention into the cytokine network
have included antagonism of pro-inflammatory
cytokines (e.g. TNF-α, IL-1, IL-6, IL-8, IL-12, IL18, IL-23) with neutralizing antibodies and soluble
receptors; application of recombinant cytokines
(e.g. IL-4, IL-10, IL-11, IFN-γ) to shift the cytokine
balance; and administration of small molecules
to modulate cytokine expression or signaling (5).
The treatment with IL-4, IL-10 and IL-11 downregulates type I cytokine pro-inflammatory pathways
in psoriasis lesions. IL-11 therapy has demonstrated
immunomodulatory activity to regulate both T cell
and macrophage function (6).
Aims of this study were to assess the safety
and efficacy of oral low-dose cytokines (IL-4, IL-10
e IL-11) in the treatment of psoriasis and to evaluate
the effects of this therapy in the quality of life.
methotrexate).
Protocol inclusion criteria concerned also patients
in more serious conditions, in order to assess whether
patients not responding to phototherapy treatment could
benefit from the studied therapy.
Study design
This is a multicenter, double-blind, randomized,
placebo-controlled clinical trial, alternated according to
a cross-over model, lasting eight months overall. Since
the beginning of the trial, for a period of 3 months, all
individuals enrolled in the study were treated with low
dose cytokines or with placebo solution. After a wash-out
period of 60 days, the administration of the experimental
product was resumed for a further three-month period,
thus implementing the cross-over experimental model.
After signing an informed consent the patients were
enrolled progressively in the different experimental sites
and identified with their initials as well as through a serial
number.
A randomization procedure performed using a tailored
software has generated two equal parts, one containing
cytokines and one containing placebo. These preparations
were marked with a serial number and an identifying code
of the three products. Each preparation was allocated to
the related subject. The chief of the Guna Labs (GUNA
Spa, Milan - Italy) was the only one aware of the product
content. A yellow label identified the products prepared
for the first period of treatment; a green label identified
the products prepared for the second period of treatment.
One group was treated with Guna-Interleukin 4, GunaInterleukin 10, Guna-Interleukin 11 at the concentration
of 10 fg/ml in a hydroalcoholic solution 30% for oral use
(GUNA Spa, Milan, Italy) administered at a dose of 20
drops twice daily, taken on an empty stomach from 8.00
to 10.00 and from 19.00 to 21.00. The other group was
treated with placebo (hydroalcoholic solution at 30%
containing no active ingredients) with the same mode of
administration. Neither the doctor nor the patient were
aware of the nature of the preparation assigned.
At the end of the first 3 months of treatment, patients
underwent a new clinical control and after a washout period of 60 days, the 2 experimental groups were
inverted (cross-over model). This in order to reduce the
possible error factors in the evaluation, as well as to give
the possibility to both groups to benefit from the therapy
(Fig. 1).
Since our primary goal was to assess the activity
of the three interleukins, the sample size was calculated
considering the possible reduction of PASI score within
treated patients by means of a crossover design. On the
other hand, from our previous experiences, we suspected
a possible long lasting carry-over effect exerted by the
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MATERIALS AND METHODS
Subjects
Forty-eight patients with moderate to slight psoriasis,
according to the psoriasis area and severity index (PASI),
were enrolled in this study, 41 (17 females and 24 males;
mean age 46.2± std.dev. 11.7 years) were evaluated. Table
I shows the patients’ epidemiological and clinical features.
Patients with guttate psoriasis, palmoplantar, pustular
and erythrodermic psoriasis and other skin diseases were
excluded.
Patients who underwent topic or systemic therapies
specific for psoriasis, respectively in 15 and 90 days prior
the enrolment.
Other exclusion criteria: subjects under 18 years of
age and subjects in therapy for other pathologies with
steroid (topical or systemic), anti-TNF-α therapies, immunosuppressive agents (e.g., cyclosporine, tacrolimus or
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interleukins (so interfering with the crossover results), and
therefore we chose a sample size as we were measuring the
PASI score in a “before-after” design. Assuming to recruit
20 subjects, in which the average reduction of PASI score
at end of protocol should have been 1.5, with a standard
deviation of reduction equal to 2, a sample of 20 patients
would have reached a 87% power (with an error α=0.05)
to detect the expected difference (before-after) within the
group starting with interleukins. Then, the same sample
size was chosen also for the other group, bearing in mind
that our focus would have been on group firstly treated
with interleukins.
Clinical assessment
Disease severity was assessed using the psoriasis area
and severity index (PASI). The PASI is a dermatological
index conventionally used, through which are evaluated
extension, erythema, infiltration and desquamation in the
areas of the head, arms, trunk and legs. The degree of severity of psoriatic manifestations provides a score ranging
from 0 (no psoriasis) to 72 (maximum value, severe psoriasis). Higher scores represent greater degrees of disease
severity (7). Severe psoriasis is defined as a PASI score
>12 (8).
For the evaluation of individual distress it was used a
Dermatology Life Quality Index (DLQI). This questionnaire fulfils reliability, validity, repeatability, and internal
consistency requirements. It is meant for adults older than
16 years. It is self-administered and consists of 10 questions and measures 6 categories: symptoms and feelings
(questions 1 and 2), daily activities (questions 3 and 4),
leisure (questions 5 and 6), work and school (question 7),
personal relationships (questions 8 and 9), and treatment
(question 10). All questions relate “to the last week.” Each
question is answered using a tick box, with not at all, a
little, a lot or very much, being scored from 0 to 3. The
DLQI is calculated by summing the scores of each question, resulting in a minimum score of 0 (no impairment) to
a maximum score of 30 (maximum impairment) in order
to measure Heath Related Quality of Life (HRQoL) in patients. Higher scores represent greater degrees of HRQoL
impairment (14). Severe impact on HRQoL by psoriasis is
defined as a DLQI >10 (9).
scores; the post-hoc test according to Conover was used
to check the pairwise comparisons. The Mann-Whitney
U test was used to evaluate the difference of the scores
between groups at baseline. Statistical significance was
assumed with p<0.05.
RESULTS
Forty-eight patients were enrolled in the study in
a period of 12 months; of these, 41 patients completed the entire experimental design and could be
evaluated (Fig. 2). 34 subjects had a PASI score at
enrollment <10; 7 had a PASI score at enrollment
<30. All the recruited patients were divided in two
groups, group A and group B. Group A, consisting
of 23 individuals, performed the experimental design taking at time T0-T3 active preparation and at
time T3-T6 the placebo. Group B, consisting of 18
subjects, conversely began with placebo in the first
time and then took the cytokines in the second time
(Fig. 1).
The global PASI score for the 41 patients at baseline (T0) ranged from a minimum of 1 to a maximum
of 21 (median=5). Considering the 2 groups separately, the PASI of group A (n=23) is 5 (1-18, IQR
2-8). In group B (n=18) the PASI at baseline (T0) is
5 ranged (1-21, IQR 2-8). No significant differences
are observed at baseline between two groups (MannWhitney U test: p>0.9999).
Globally, in the 41 evaluated patients, who completed the study, it is observed a PASI significant
reduction (Friedman test: p=0.0096). According to
the Conover’s post-hoc test, significant difference
(p<0.05) is evident between values at time T0 and at
time T3 and between values at time T0 and at time
T6, but not between T3 and T6 time values (Table
II).
Group A (n=23), treated during the first period,
shows a PASI significant reduction (Friedman test:
p=0.0275) (Table III). A significant difference according to Conover (p<0.05) was found only between values at T0 and at T6, whereas variations at
T3 are not significant.
In group B (n=18) no significant differences
between values at T0 and at T3 and between values at T0 and at T6 are observed (Friedman test:
p=0.23348) (Table III).
The DLQI for all patients (n=41) at baseline is 5
(0-23, IQR 2-10) (Table II). In group A, the DLQI
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Statistical analysis
All data were analyzed with usual descriptive
statistics. D’Agostino-Pearson test, together with visual
inspection of the histogram, were used to check the
variable distributions: mean and standard deviation were
used for the normally distributed variables, while median
and IQR (InterQuartile Range) were used for non-normal
ones. The Friedman test was used to analyze the withingroup repeated measure variation of both PASI and DLQI
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M.L. ROBERTI ET AL.
Table I. Epidemiological and clinical features of patients with psoriasis
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Fig. 1. Study design
is 4 (0-14, IQR 1.25-10) (Table III). In group B, the
DLQI is 6 (0-23, IQR 3-10) (Table IV). The DLQI at
baseline did not show any difference between the 2
groups (Mann-Whitney U test: p=0.2688).
The DLQI decreased significantly in all subjects
(n=41) compared to baseline (p<0.0001). A
significant difference according to Conover (p<0.05)
was observed between values at T0 and at T3 and
between values at T0 and at T6, but not between T3
and T6 (Table II).
In group A (n=23) we observed a DLQI significant
reduction (p=0.0134) between values at T0 and at
T6. A significant pairwise difference (p<0.05) was
found between baseline and values at 6 months, but
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Fig. 2. Flow chart of enrolled patients.
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Table II. The PASI and DLQI in all subjects. Data are expressed in median with range (minimum and maximum).
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Table III. The PASI and DLQI in group A subjects. Data are expressed in median with range (minimum and maximum).
Table IV. The PASI and DLQI in group B subjects. Data are expressed in median with range (minimum and maximum).
not between baseline and values at 3 months, nor
between 3 and 6 months (Table III).
In group B (n=18) the variation is still significant
(p=0.0005) and the significant difference is kept
between baseline and 3 months and between baseline
and 6 months (Table IV).
DISCUSSION
In this study we demonstrated that therapy with
low dose of cytokines (IL-4, IL-10 and IL-11) was
safe and reduced severity of psoriasis vulgaris. In addition, this therapy improved the quality of life.
No adverse event was reported during the study
in patients with psoriasis vulgaris. At our knowledge
this is the first study who demonstrated the safety of
low dose cytokine.
In this study the low dose cytokine ameliorate
the PASI score significantly (p=0.00960) between
baseline and 3 months and between baseline and 6
months. These results confirmed the hypothesis that
anti-inflammatory cytokines (IL-4, IL-10, IL-11)
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M.L. ROBERTI ET AL.
showed a key role in the pathogenesis of psoriasis. A
network of pro-inflammatory cytokines is a central
feature in the pathophysiology of cutaneous inflammatory diseases. While anti-TNF therapeutics have
proven to be effective for the treatment of psoriasis,
clinical investigations have now begun with other
cytokine-directed therapies, such as those targeting
IFN-γ, IL-12, and IL-18.
Since the IL-4 and IL-10 have reduced psoriatic
plaques, we can suppose that the administration of
these low dose cytokines restores the normal balance
of cytokine network (10).
Ghoreschi et al. demonstrated that in patients
with psoriasis IL-4 therapy was well tolerated and
within six weeks all patients showed decreased clinical scores. IL-4 therapy can induce Th2 differentiation in human CD4+ T cells and is promising as a
potential treatment for psoriasis (11).
IL-10 is an important immunoregulatory cytokine
produced by many cell populations. Its main biological function seem to be the limitation and termination of inflammatory responses and the regulation of
differentiation and proliferation of several immune
cells such as T cells, B cells, natural killers, antigenpresenting cells, mast cells, and granulocytes. Recombinant human IL-10 has been produced and is
currently being tested in clinical trials. This includes
rheumatoid arthritis, inflammatory bowel disease,
psoriasis, organ transplantation, and chronic hepatitis C (12, 13). A study involving 28 patients with
moderate-to severe psoriasis showed that treatment
with rhIL-10 resulted in only temporary clinical improvement in psoriasis, despite sustained systemic
decreases in pro-inflammatory and type 1 cytokine
production (14).
IL-11, a multifunctional cytokine, modulates
macrophage and type 1 T-lymphocyte function in
cell culture and shows anti-inflammatory activity
in animal models. Trepicchio et al. evaluated the effect of subcutaneous delivery of rhIL-11 in patients
with psoriasis. Seven of 12 patients responded well
to rhIL-11 treatment. Amelioration of disease by
rhIL-11, as shown by reduced keratinocyte proliferation and cutaneous inflammation, was associated
with decreased expression of products of diseaserelated genes, including K16, iNOS, IFN-γ, IL-8,
IL-12, TNF-α, IL-1β, and CD8+, and with increased
expression of endogenous IL-11 (6). The ability of
rhIL-11 to modulate cytokine production from activated CD4+ T cells provides a mechanism through
which rhIL-11 may ameliorate such inflammatory
diseases as psoriasis (15). Conversely, in comparison with normal controls, bone marrow stromal cells
from patients with psoriasis showed no alteration in
the levels of GM-CSF, IL-11, IL-7 (16).
Our data confirm that most patients with psoriasis
have poor quality of life prior to treatment, and that
improvement in psoriasis lesion and symptoms (e.g.
pruritus, restlessness) with treatment significantly
correlates with improvement in DLQI.
We can conclude that low dose cytokines are safe
and effective in the treatment of psoriasis vulgaris.
Considering the initial suspect of carry over effect,
the results of this exploratory study seemed to
highlight a long time action of interleukins. The
latter is shown also after stopping the treatment.
Further trials with a longer observational period are
necessary to confirm our preliminary data.
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AKNOWLEDGEMENTS
We wish to thank M.Assirati, B.Fiorentino,
A.Crippa, A Nazari, B.Arsuffi, G.Tomasini,
M.Passafaro, S.Cangemi, R.Malvicini, M.Siffredi,
C.Gremita, F.Mazzola, L.Morlando, L.Cendret for
their valuable clinical contribution to the achievement
of this work. We wish to thank V.Miranda (Clinical
Research Unit Guna S.p.a Milan-Italy) for his
helpful scientific discussion and critical revision
of the manuscript. We also thank M.Nichelatti
and C.Mattana for the bioinformatic analysis,
M.Campanella (Clinical Research Unit Guna S.p.a
Milan-Italy) for her scientific and technical support
in data management, F.Melzi (Guna S.p.a) for her
linguistic assistance.
REFERENCES
1. Kurd SK, Gelfand JM. The prevalence of previously
diagnosed and undiagnosed psoriasis in US adults:
results from NHANES 2003-2004. J Am Acad Dermatol 2009; 60:218-24.
2. Krueger GG, Duvic M. Epidemiology of psoriasis:
clinical issues. J. Invest Dermatol 1994; 102:14-18S.
3. Gudjonsson JE, Johnston A, Sigmundsdottir H et al.
Journal of Biological Regulators & Homeostatic Agents
4.
5.
6.
7.
8.
9.
10.
Immunopathogenic mechanisms in psoriasis. Clin
Exp Immunol 2004; 135:1-8.
Nestle FO, Kaplan DH, Barker J. Psoriasis. N Engl J
Med 2009; 30(361):496-509.
Numerof RP, Asadullah K. Cytokine and anti-cytokine therapies for psoriasis and atopic dermatitis.
BioDrugs 2006; 20:93-103.
Trepicchio WL, Ozawa M, Walters IB. Interleukin-11
therapy selectively downregulates type I cytokine
proinflammatory pathways in psoriasis lesions. J Clin
Invest 1999; 104:1527-37.
Fredriksson T, Petersson U. Severe psoriasis oral
therapy with a new retinoid. Dermatologica 1978;
157: 238-44.
Schmitt J, Wozel G. The Psoriasis Area and Severity
Index Is the Adequate Criterion to Define Severity in
Chronic Plaque-Type Psoriasis. Dermatology 2005;
210:194-9
Finlay AY, Khan GK. Dermatology Life Quality Index (DLQI)-a simple practical measure for routine
clinical use. Clin Exp Dermatol 1994; 19:210-6.
Numerof RP, Dinarello CA, Asadullah K. Cytokines
as potential therapeutic targets for inflammatory skin
diseases. Eur Cytokine Netw 2005; 16:101-3.
11. Ghoreschi K, Thomas P, Breit S, Dugas M, Mailhammer R, van Eden W, et al. Interleukin-4 therapy
of psoriasis induces Th2 responses and improves human autoimmune disease. Nat Med 2003; 9:40.
12. Asadullah K, Sterry W, Volk HD. Interleukin-10
therapy – review of a new approach. Pharmacol Rev
2003; 55:241.
13. Asadullah K, Sabat R, Friedrich M, Volk HD, Sterry
W. Interleukin-10: an important immunoregulatory
cytokine with major impact on psoriasis. Curr Drug
Targets Inflamm Allergy 2004; 3:185-92.
14. Kimball AB, Kawamura T, Tejura K, et al. Clinical
and immunologic assessment of patients with psoriasis in a randomized, double-blind, placebo-controlled trial using recombinant human interleukin 10.
Arch Dermatol 2002; 138:41.
15.���������������������������������������������������
Bozza M, Bliss JL, Dorner AJ, Trepicchio WL. Inter������
leukin-11 modulates Th1/Th2 cytokine production
from activated CD4+ T cells. J Interferon Cytokine
Res 2001; 21:21-30.
16. Zhang K, Liu R, Yin G, Li X, Li J, Zhang J. Differential cytokine secretion of cultured bone marrow
stromal cells from patients with psoriasis and healthy
volunteers. Eur J Dermatol 2010; 20:49-53.
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